Muscle invasive bladder cancer (MIBC) is often lethal and non-MIBC (NMIBC) can recur and progress, yet prognostic markers are currently inadequate. SAM68, a member of RNA-binding proteins, has been reported to contribute to progression of other cancers.
Zhang et al BMC Cancer (2015) 15:364 DOI 10.1186/s12885-015-1367-x RESEARCH ARTICLE Open Access Utility of SAM68 in the progression and prognosis for bladder cancer Zhiling Zhang1,2,3†, Chunping Yu4†, Yonghong Li1,2,3, Lijuan Jiang1,2,3 and Fangjian Zhou1,2,3* Abstract Background: Muscle invasive bladder cancer (MIBC) is often lethal and non-MIBC (NMIBC) can recur and progress, yet prognostic markers are currently inadequate SAM68, a member of RNA-binding proteins, has been reported to contribute to progression of other cancers The aim of this study is to investigate the potential utility of SAM68 in the progression and prognosis of bladder cancer Methods: Quantitative PCR and immunohistochemistry were utilized to examine the expression of SAM68 in ten pairs of MIBC and adjacent normal bladder urothelium, and eight pairs of MIBC and non-MIBC (NMIBC) tissues from the same patient Moreover, SAM68 protein expression level and localization were examined by immunohistochemistry in 129 clinicopathologically characterized MIBC samples Prognostic associations were determined by multivariable analysis incorporating standard prognostic factors Results: SAM68 expression was elevated in MIBC tissues compared with adjacent normal bladder urothelium, and was increased at both transcriptional and translational levels in MIBC tissues compared with NMIBC tissues of the same patient For MIBC, high expression and nucleus-cytoplasm co-expression of SAM68 were associated with higher T-stage, higher N-stage and worse recurrence-free survival Five-year recurrence-free survival was 80% and 52.9% for MIBC patients with low and high SAM68 expression, respectively (p = 0.001) SAM68 nucleus-cytoplasm co-expression associated with worse 5-year recurrence-free survival rate (49.2%) than SAM68 expression confined to the nucleus (82.5%) or cytoplasm (75.5%) alone On multivariable analysis SAM68 expression level, SAM68 nucleus-cytoplasm co-expression, T-stage, and N-stage were all independent prognostic factors for recurrence-free survival of MIBC patients Conclusions: SAM68 expression is increased in MIBC when compared to normal urothelium and NMIBC, and appears to be a potentially useful prognostic marker for MIBC Keywords: SAM68, Bladder cancer, Prognosis, Biomarker, Progression Background Bladder cancer is the fourth most-common malignancy in men, accounting for 7% of all cancer cases In 2014 there will be 74,690 new cases of bladder cancer in the USA alone, leading to 15,580 cancer-related deaths [1] About 25% of bladder cancers are muscle invasive (MIBC) at presentation [2,3], and such patients are at high risk of cancer death even after intensive treatment, with a five-year survival rate of 33% and 5.4% for regional and distal metastasis MIBC, respectively [4] * Correspondence: zhoufj@sysucc.org.cn † Equal contributors State Key Laboratory of Oncology in Southern China, Guangzhou, China Department of Urology, Sun Yat-sen University Cancer Center, Guangzhou, China Full list of author information is available at the end of the article Approximately 1/3 patients have cancer recurrence after cystectomy even with adjuvant chemotherapy [5] Although altered expression of oncogenes, tumor suppressors, and other markers have been found in bladder cancer [6], the prognostic utility of currently available biomarkers remains limited, and they are not routinely incorporated into clinical practice The identification of novel biomarkers involved in the progression of bladder cancer would greatly facilitate patient management SAM68 (Src-associated in mitosis, 68 kDa) is a member of the STAR (signal transduction and activation of RNA) family of RNA-binding proteins [7], and has shown potential as a biomarker for malignancy [8-11] SAM68 proteins have an hnRNP K homology domain (KH domain) that locates within a larger GSG © 2015 Zhang et al.; licensee BioMed Central This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Zhang et al BMC Cancer (2015) 15:364 (GRP33-SAM68-GLD1) domain that is required for specificity and high-affinity binding to RNA [7,12] This multimodular structure allows SAM68 protein to exert different functions in the cell, including regulation of the cell cycle, proliferation, and apoptosis SAM68 has been reported to associate with progression and/or prognosis for a variety of malignancies, including renal cell carcinoma [8], prostate cancer [9], cervical cancer [10] and breast cancer [11] For example, increased expression and cytoplasmic localization of SAM68 correlated with clinical outcomes and prognosis for patients with breast cancer An in vitro study revealed that down-regulation of SAM68 in breast cancer cells inhibited cell proliferation by blocking the transition from G1 to S phase, and the Akt/GSK-3β signaling and FOXO/p21/p27 pathway appeared to be involved [11] In early-stage cervical cancer, increased expression of SAM68 associated with lymph node metastasis apparently by promoting cellular motility and invasion, again through the Akt/ GSK-3β pathway [10] In the current study, we explore the potential utility of SAM68 expression and localization in human bladder cancer, and report correlations with clinical outcomes, progression and prognosis Methods Patients and tissue specimens Patient consent and approval from the Sun Yat-sen University Cancer Center Institutional Review Board were obtained for the use of these clinical materials for research purposes Ten pairs of MIBC tissue specimens and corresponding non-tumorous specimens were obtained from patients with bladder cancer who underwent radical cystectomy at the Cancer Center of the Sun Yat-sen University (Guangzhou, P R China) Eight paired of non-muscle invasive (NMIBC) and MIBC tissues from the same patient were obtained from TURBT and radical cystectomy, respectively All excised tissues were obtained within h after surgery and were immediately placed in liquid nitrogen until further analysis Immunohistochemistry analyses were performed on 129 paraffinembedded radical cystectomy samples, which were histologically diagnosed as MIBC at the Cancer Center, Sun Yat-sen University, between 2000 and 2008 Tumornode-metastasis (TNM) staging was determined according to the 2010 American Joint Committee on Cancer TNM classification of bladder cancer [13] The detail of patients’ information are summarized in Table The median follow-up period for this cohort of patients was 32 months (range, 6-104 months) During the follow-up period, 35 patients had tumor recurrence RNA extraction and quantitative PCR Total RNA from tumor and adjacent non-tumorous tissues was extracted using the TRIzol reagent (Invitrogen) Page of according to the manufacturer's instructions Quantitative polymerase chain reaction (PCR) was performed according to standard methods as described previously [8] PCR primers and probes were designed with the use of Primer Express Software v.2.0 (Applied Biosystems) as described previously [8] Immunohistochemistry Immunohistochemistry (IHC) was performed to study altered SAM68 protein expression levels in 129 human MIBC tissues, as well as the ten pairs of MIBC tissue specimens and corresponding non-tumorous specimens, and eight paired of NMIBC and MIBC tissues In brief, μm-thick tissue sections were incubated with polyclonal rabbit antibody against SAM68 (1:200; Abgent) at 4°C overnight Before incubation with the primary antibody, the sections were treated for antigen retrieval with ethylene diamine tetraacetic acid buffer followed by heating in a microwave oven For negative controls, the rabbit anti-SAM68 antibody was restored with normal nonimmune serum After washing, tissue pieces were treated with biotinylated anti-rabbit secondary antibody (Zymed), followed by further incubation with streptavidin -horseradish peroxidase complex (Zymed) Tissue sections were then immersed in 3,3′-diaminobenzidine and counterstained with 10% Mayer's hematoxylin, dehydrated, and mounted The degree of immunostaining of paraffin-embedded sections was reviewed and scored independently by two observers based on the proportion of positively-stained tumor cells and the intensity of staining The method has been introduced in detail previously [8] The staining index was calculated as the product of the staining intensity score and the proportion of positive tumor cells Using this method of assessment, we evaluated SAM68 expression in MIBC tissues by determining the staining index, with scores of 0, 1, 2, 3, 4, 6, 8, 9, and 12 SAM68 threshold values were chosen on the basis of a measure of heterogeneity with the logrank test statistical analysis with respect to recurrence-free survival An optimal threshold value was identified: a staining index score >6 was considered to show high SAM68 expression, whereas a staining index score